ABSTRACT The overall goal of this project is to engage undergraduate and graduate students in innovative studies in molecular toxinology that have the potential to contribute to important advances in our understanding of snake venom pathology and the development of novel antibody-based therapeutics. According to WHO statistics, it is estimated that in 2020 there will be 125,000 new deaths world-wide reported due to snake envenomation. In spite of its importance for snake bite envenomation as a neglected tropical disease and global health issue, very little is known of the mechanism of action of snake venom and intrinsic toxic/lethal contribution. The scientific foundation for our study is that a specific combination of snake venom toxins plays a critical role in snakebites by causing necrosis, coagulopathy, severe hemorrhaging and eventually death. Inasmuch as individual toxins have been evaluated, synergistic mechanism for snake venom pathology are lacking and warranted for antidote development. We hypothesize that precise toxicity evaluation and snake venom analytics of crude/fractionated venom and recombinant toxins will provide a model in which priority can be given to the most toxic venom component(s). To test our hypothesis, we will address (3) Specific Aims: Specific Aim 1: To profile snake venom composition, combinational assessment and intrinsic toxicity. We will purify and characterize snake venom of Crotalid snake species. Characterization will include profiling snake venom to identify the toxins in each fraction and abundance. Students will investigate the mechanism of action different combinations of venom fractions. We will characterize the lethal concentrations/doses of each venom fraction on HUVEC cells, blood coagulation, and will validate the toxicity score for each fraction using an in vivo lethality mouse model. Specific Aim 2: To produce recombinant venom peptides. We will clone these toxins from our collection of cDNA libraries and will generate plasmids and transform competent E.coli cells for protein expression and purification. Development of methods to produce recombinant toxins is essential for antivenom development and investigating toxicity and targets for medically relevant venom peptide. These studies will also be essential to use toxins in solving structures to venom toxins or structures in complexes with known targets. Specific Aim 3: To identify snake venom biomarkers in plasma exosomes. We will explore the ?venom-reactome? using novel Evtrap technology and proteomics in discovery-based analysis of snake venom biomarkers from mouse plasma exosomes. Comprehensive snake venom biomarker analysis will shed new light on the pathophysiology, metabolic, biological, cellular and immunological responses to snake venom.